Virus-Host Interactions Drive Contrasting Bacterial Diel Dynamics in the Ocean.

Marine organisms perform a sea of diel rhythmicity. Planktonic diel dynamics have been shown to be driven by light, energy resources, circadian rhythms, and the coordinated coupling of photoautotrophs and heterotrophic bacterioplankton. Here, we explore the diel fluctuation of viral production and decay and their impact on the total and active bacterial community in the coastal and open seawaters of the South China Sea. The results showed that the night-production diel pattern of lytic viral production was concurrent with the lower viral decay at night, contributing to the accumulation of the viral population size during the night for surface waters. The diel variations in bacterial activity, community composition, and diversity were found highly affected by viral dynamics. This was revealed by the finding that bacterial community diversity was positively correlated to lytic viral production in the euphotic zone of the open ocean but was negatively related to lysogenic viral production in the coastal ocean. Such distinct but contrasting correlations suggest that viral life strategies can not only contribute to diversifying bacterial community but also potentially piggyback their host to dominate bacterial community, suggesting the tightly synchronized depth-dependent and habitat-specific diel patterns of virus-host interactions. It further implies that viruses serve as an ecologically important driver of bacterial diel dynamics across the ocean, highlighting the viral roles in bacterial ecological and biogeochemical processes in the ocean.

The smallest in the deepest: the enigmatic role of viruses in the deep biosphere.

It is commonly recognized that viruses control the composition, metabolism, and evolutionary trajectories of prokaryotic communities, with resulting vital feedback on ecosystem functioning and nutrient cycling in a wide range of ecosystems. Although the deep biosphere has been estimated to be the largest reservoir for viruses and their prokaryotic hosts, the biology and ecology of viruses therein remain poorly understood. The deep virosphere is an enigmatic field of study in which many critical questions are still to be answered. Is the deep virosphere simply a repository for deeply preserved, non-functioning virus particles? Or are deep viruses infectious agents that can readily infect suitable hosts and subsequently shape microbial populations and nutrient cycling? Can the cellular content released by viral lysis, and even the organic structures of virions themselves, serve as the source of bioavailable nutrients for microbial activity in the deep biosphere as in other ecosystems? In this review, we synthesize our current knowledge of viruses in the deep biosphere and seek to identify topics with the potential for substantial discoveries in the future.

Reduced bacterial mortality and enhanced viral productivity during sinking in the ocean.

Particle sinking is an important process in the ocean, influencing the biogeochemical cycle and driving the long-term preservation of carbon into the deep sea via the biological pump. However, as an important component of marine ecosystems, the role of viruses during sinking is still poorly understood. Therefore, we performed a series of transplantation experiments in the South China Sea to simulate environmental changes during sinking and investigate their effects on viral eco-dynamics and life strategy. Our study demonstrated increased viral production but decreased virus-mediated bacterial mortality after transplantation. A larger burst size and switch from the lysogenic to lytic strategy were shown to contribute to enhanced viral productivity. We provide experimental evidence that surface viral ecological characteristics changed dramatically after transplantation into deep-sea waters, indicating a potential importance of viruses during vertical sinking in the ocean. This effect probably provides positive feedback on the efficiency of the biological pump.

Aquatic Viruses and Climate Change.

The viral component in aquatic systems clearly needs to be incorporated into future ocean and inland water climate models. Viruses have the potential to influence carbon and nutrient cycling in aquatic ecosystems significantly. Changing climate likely has both direct and indirect influence on virus-mediated processes, among them an impact on food webs, biogeochemical cycles and on the overall metabolic performance of whole ecosystems. Here we synthesise current knowledge on potential climate-related consequences for viral assemblages, virus-host interactions and virus functions, and in turn, viral processes contributing to climate change. There is a need to increase the accuracy of predictions of climate change impacts on virus- driven processes, particularly of those linked to biological production and biogeochemical cycles. Comprehension of the relationships between microbial/viral processes and global phenomena is essential to predict the influence on as well as the response of the biosphere to global change.

Viral Lysis Alters the Optical Properties and Biological Availability of Dissolved Organic Matter Derived from Prochlorococcus Picocyanobacteria.

Phytoplankton contribute almost half of the world's total primary production. The exudates and viral lysates of phytoplankton are two important forms of dissolved organic matter (DOM) in aquatic environments and fuel heterotrophic prokaryotic metabolism. However, the effect of viral infection on the composition and biological availability of phytoplankton-released DOM is poorly understood. Here, we investigated the optical characteristics and microbial utilization of the exudates and viral lysates of the ecologically important unicellular picophytoplankton Prochlorococcus Our results showed that Prochlorococcus DOM produced by viral lysis (Pro-vDOM) with phages of three different morphotypes (myovirus P-HM2, siphovirus P-HS2, and podovirus P-SSP7) had higher humic-like fluorescence intensities, lower absorption coefficients, and higher spectral slopes than DOM exuded by Prochlorococcus (Pro-exudate). The results indicate that viral infection altered the composition of Prochlorococcus-derived DOM and might contribute to the pool of oceanic humic-like DOM. Incubation with Pro-vDOM resulted in a greater dissolved organic carbon (DOC) degradation rate and lower absorption spectral slope and heterotrophic bacterial growth rate than incubation with Pro-exudate, suggesting that Pro-vDOM was more bioavailable than Pro-exudate. In addition, the stimulated microbial community succession trajectories were significantly different between the Pro-exudate and Pro-vDOM treatments, indicating that viral lysates play an important role in shaping the heterotrophic bacterial community. Our study demonstrated that viral lysis altered the chemical composition and biological availability of DOM derived from Prochlorococcus, which is the numerically dominant phytoplankton in the oligotrophic ocean.IMPORTANCE The unicellular picocyanobacterium Prochlorococcus is the numerically dominant phytoplankton in the oligotrophic ocean, contributing to the vast majority of marine primary production. Prochlorococcus releases a significant fraction of fixed organic matter into the surrounding environment and supports a vital portion of heterotrophic bacterial activity. Viral lysis is an important biomass loss process of Prochlorococcus However, little is known about whether and how viral lysis affects Prochlorococcus-released dissolved organic matter (DOM). Our paper shows that viral infection alters the optical properties (such as the absorption coefficients, spectral slopes, and fluorescence intensities) of released DOM and might contribute to a humic-like DOM pool and carbon sequestration in the ocean. Meanwhile, viral lysis also releases various intracellular labile DOM, including amino acids, protein-like DOM, and lower-molecular-weight DOM, increases the bioavailability of DOM, and shapes the successive trajectory of the heterotrophic bacterial community. Our study highlights the importance of viruses in impacting the DOM quality in the ocean.

Viral control of biomass and diversity of bacterioplankton in the deep sea.

Viral abundance in deep-sea environments is high. However, the biological, ecological and biogeochemical roles of viruses in the deep sea are under debate. In the present study, microcosm incubations of deep-sea bacterioplankton (2,000 m deep) with normal and reduced pressure of viral lysis were conducted in the western Pacific Ocean. We observed a negative effect of viruses on prokaryotic abundance, indicating the top-down control of bacterioplankton by virioplankton in the deep-sea. The decreased bacterial diversity and a different bacterial community structure with diluted viruses indicate that viruses are sustaining a diverse microbial community in deep-sea environments. Network analysis showed that relieving viral pressure decreased the complexity and clustering coefficients but increased the proportion of positive correlations for the potentially active bacterial community, which suggests that viruses impact deep-sea bacterioplankton interactions. Our study provides experimental evidences of the crucial role of viruses in microbial ecology and biogeochemistry in deep-sea ecosystems.

Effects of elevated pCO2 and feeding on net calcification and energy budget of the Mediterranean cold-water coral Madrepora oculata.

Ocean acidification is a major threat to calcifying marine organisms such as deep-sea cold-water corals (CWCs), but related knowledge is scarce. The aragonite saturation threshold (Ωa) for calcification, respiration and organic matter fluxes were investigated experimentally in the Mediterranean Madrepora oculata Over 10 weeks, colonies were maintained under two feeding regimes (uptake of 36.75 and 7.46 µmol C polyp-1 week-1) and exposed in 2 week intervals to a consecutively changing air-CO2 mix (pCO2) of 400, 1600, 800, 2000 and 400 ppm. There was a significant effect of feeding on calcification at initial ambient pCO2, while with consecutive pCO2 treatments, feeding had no effect on calcification. Respiration was not significantly affected by feeding or pCO2 levels. Coral skeletons started to dissolve at an average Ωa threshold of 0.92, but recovered and started to calcify again at Ωa≥1. The surplus energy required to counteract dissolution at elevated pCO2 (≥1600 µatm) was twice that at ambient pCO2 Yet, feeding had no mitigating effect at increasing pCO2 levels. This could be due to the fact that the energy required for calcification is a small fraction (1-3%) of the total metabolic energy demand and corals even under low food conditions might therefore still be able to allocate this small portion of energy to calcification. The response and resistance to ocean acidification are consequently not controlled by feeding in this species, but more likely by chemical reactions at the site of calcification and exchange processes between the calicoblastic layer and ambient seawater.

Colonization and release processes of viruses and prokaryotes on artificial marine macroaggregates.

Marine organic aggregates are sites of high of viral accumulation; however, still little is known about their colonization processes and interactions with their local bacterial hosts. By taking advantage of a novel approach (paramagnetic functionalized microsphere method) to create and incubate artificial macroaggregates, we examined the small-scale movements of viruses and bacteria between such marine snow particles and the surrounding water. The examination of the codynamics of both free-living and attached viral and bacterial abundance, over 12 hours of incubation in virus-free water, suggests that aggregates are rather comparable to viral factories than to viral traps where a significant part of the virions production might be locally diverted to the water column. Also, the near-zero proportion of lysogenized cells measured in aggregates after mitomycin-C induction seems to indicate that lysogeny is not a prominent viral reproduction pathway in organic aggregates where most viruses might rather be virulent. Finally, we hypothesize that, contrary to bacteria, which can use both strong attachment and detachment from aggregates (two-way motion of bacteria), the adsorption of planktonic viruses appears to be numerically negligible compared to their massive export from the aggregates into the water column (one-way motion of viruses).

Comparison of deep-water viromes from the atlantic ocean and the mediterranean sea.

The aim of this study was to compare the composition of two deep-sea viral communities obtained from the Romanche Fracture Zone in the Atlantic Ocean (collected at 5200 m depth) and the southwest Mediterranean Sea (from 2400 m depth) using a pyro-sequencing approach. The results are based on 18.7% and 6.9% of the sequences obtained from the Atlantic Ocean and the Mediterranean Sea, respectively, with hits to genomes in the non-redundant viral RefSeq database. The identifiable richness and relative abundance in both viromes were dominated by archaeal and bacterial viruses accounting for 92.3% of the relative abundance in the Atlantic Ocean and for 83.6% in the Mediterranean Sea. Despite characteristic differences in hydrographic features between the sampling sites in the Atlantic Ocean and the Mediterranean Sea, 440 virus genomes were found in both viromes. An additional 431 virus genomes were identified in the Atlantic Ocean and 75 virus genomes were only found in the Mediterranean Sea. The results indicate that the rather contrasting deep-sea environments of the Atlantic Ocean and the Mediterranean Sea share a common core set of virus types constituting the majority of both virus communities in terms of relative abundance (Atlantic Ocean: 81.4%; Mediterranean Sea: 88.7%).

Interactive effects of viral and bacterial production on marine bacterial diversity.

A general model of species diversity predicts that the latter is maximized when productivity and disturbance are balanced. Based on this model, we hypothesized that the response of bacterial diversity to the ratio of viral to bacterial production (VP/BP) would be dome-shaped. In order to test this hypothesis, we obtained data on changes in bacterial communities (determined by terminal restriction fragment length polymorphism of 16S rRNA gene) along a wide VP/BP gradient (more than two orders of magnitude), using seawater incubations from NW Mediterranean surface waters, i.e., control and treatments with additions of phosphate, viruses, or both. In December, one dominant Operational Taxonomic Unit accounted for the major fraction of total amplified DNA in the phosphate addition treatment (75±20%, ± S.D.), but its contribution was low in the phosphate and virus addition treatment (23±19%), indicating that viruses prevented the prevalence of taxa that were competitively superior in phosphate-replete conditions. In contrast, in February, the single taxon predominance in the community was held in the phosphate addition treatment even with addition of viruses. We observed statistically robust dome-shaped response patterns of bacterial diversity to VP/BP, with significantly high bacterial diversity at intermediate VP/BP. This was consistent with our model-based hypothesis, indicating that bacterial production and viral-induced mortality interactively affect bacterial diversity in seawater.

End of the century pCO2 levels do not impact calcification in Mediterranean cold-water corals.

Ocean acidification caused by anthropogenic uptake of CO2 is perceived to be a major threat to calcifying organisms. Cold-water corals were thought to be strongly affected by a decrease in ocean pH due to their abundance in deep and cold waters which, in contrast to tropical coral reef waters, will soon become corrosive to calcium carbonate. Calcification rates of two Mediterranean cold-water coral species, Lophelia pertusa and Madrepora oculata, were measured under variable partial pressure of CO2 (pCO2) that ranged between 380 µatm for present-day conditions and 930 µatm for the end of the century. The present study addressed both short- and long-term responses by repeatedly determining calcification rates on the same specimens over a period of 9 months. Besides studying the direct, short-term response to elevated pCO2 levels, the study aimed to elucidate the potential for acclimation of calcification of cold-water corals to ocean acidification. Net calcification of both species was unaffected by the levels of pCO2 investigated and revealed no short-term shock and, therefore, no long-term acclimation in calcification to changes in the carbonate chemistry. There was an effect of time during repeated experiments with increasing net calcification rates for both species, however, as this pattern was found in all treatments, there is no indication that acclimation of calcification to ocean acidification occurred. The use of controls (initial and ambient net calcification rates) indicated that this increase was not caused by acclimation in calcification response to higher pCO2. An extrapolation of these data suggests that calcification of these two cold-water corals will not be affected by the pCO2 level projected at the end of the century.

Response of bacterioplankton to a glucose gradient in the absence of lysis and grazing.

Bacterial utilization of dissolved organic matter plays an important role in marine carbon cycling. In this study, the response of bacterioplankton to a gradient of carbon (glucose) addition was investigated experimentally in a subtropical coastal environment in the absence of top-down control by viruses and flagellates. Bacterial abundance and production were stimulated by glucose addition corresponding to a gradient of glucose. Differences in the extent of stimulation suggested different bacterial life strategies under different nutrient conditions. Bacterial community diversity as revealed by denaturing gradient gel electrophoresis (DGGE) showed a unimodal productivity-diversity (number of DGGE bands) relationship after 3-day incubation. DNA fingerprinting profiling and cluster analysis showed clear and gradual changes in bacterial community structure along the gradient of glucose concentrations, reflecting the competition for carbon supply among bacterial groups. Sequencing analysis of the DGGE bands disclosed the relative abundance of seven bacterial genotypes in the Alteromonadaceae and Roseovarius that gradually decreased with the glucose enrichment while two Vibrio genotypes showed the reverse increasing trend. This suggested that Vibrio was a more successful opportunist at high carbon availability.

Effects of sodium azide on the abundance of prokaryotes and viruses in marine samples.

Flow cytometry is set to become the standard method for enumerating prokaryotes and viruses in marine samples. However, the samples need to be flash-frozen in liquid nitrogen directly after aldehyde fixation. Because liquid nitrogen may not always be available, we tested the potential of sodium azide as a preservative for prokaryotes and viruses in marine samples as a possible alternative. For that we conducted incubation experiments with untreated and sodium azide treated marine water samples at 4°C and room temperature. The data indicate that sodium azide cannot be used to maintain marine samples used for the enumeration of prokaryotes and viruses.

Development of phoH as a novel signature gene for assessing marine phage diversity.

Phages play a key role in the marine environment by regulating the transfer of energy between trophic levels and influencing global carbon and nutrient cycles. The diversity of marine phage communities remains difficult to characterize because of the lack of a signature gene common to all phages. Recent studies have demonstrated the presence of host-derived auxiliary metabolic genes in phage genomes, such as those belonging to the Pho regulon, which regulates phosphate uptake and metabolism under low-phosphate conditions. Among the completely sequenced phage genomes in GenBank, this study identified Pho regulon genes in nearly 40% of the marine phage genomes, while only 4% of nonmarine phage genomes contained these genes. While several Pho regulon genes were identified, phoH was the most prevalent, appearing in 42 out of 602 completely sequenced phage genomes. Phylogenetic analysis demonstrated that phage phoH sequences formed a cluster distinct from those of their bacterial hosts. PCR primers designed to amplify a region of the phoH gene were used to determine the diversity of phage phoH sequences throughout a depth profile in the Sargasso Sea and at six locations worldwide. phoH was present at all sites examined, and a high diversity of phoH sequences was recovered. Most phoH sequences belonged to clusters without any cultured representatives. Each depth and geographic location had a distinct phoH composition, although most phoH clusters were recovered from multiple sites. Overall, phoH is an effective signature gene for examining phage diversity in the marine environment.

Randomly amplified polymorphic DNA reveals tight links between viruses and microbes in the bathypelagic zone of the Northwestern Mediterranean Sea.

The study site located in the Mediterranean Sea was visited eight times in 2005 and 2006 to collect samples from the epipelagic (5 m), mesopelagic (200 m, 600 m), and bathypelagic (1,000 m, 2,000 m) zones. Randomly amplified polymorphic DNA PCR (RAPD-PCR) analysis was used to obtain fingerprints from microbial and viral size fractions using two different primers each. Depending on the primer used, the number of bands in the water column varied between 12 to 24 and 6 to 19 for the microbial size fraction and between 16 to 26 and 8 to 22 for the viral size fraction. The majority of sequences from the microbial fraction was related to Alphaproteobacteria, Cyanobacteria, Gammaproteobacteria, Firmicutes, and Eukaryota. Only 9% of sequences obtained from the viral fraction were of identifiable viral origin; however, 76% of sequences had no close relatives in the nr database of GenBank. Only 20.1% of complete phage genomes tested in silico resulted in potential RAPD-PCR products, and only 12% of these were targeted by both primers. Also, in silico analysis indicated that RAPD-PCR profiles obtained by the two different primers are largely representative of two different subsets of the viral community. Also, correlation analyses and Mantel tests indicate that the links between changes in the microbial and viral community were strongest in the bathypelagic. Thus, these results suggest a strong codevelopment of virus and host communities in deep waters. The data also indicate that virus communities in the bathypelagic zone can exhibit substantial temporal dynamics.

Association of marine viral and bacterial communities with reference black carbon particles under experimental conditions: an analysis with scanning electron, epifluorescence and confocal laser scanning microscopy.

Black carbon (BC), the product of incomplete combustion of fossil fuels and biomass, constitutes a significant fraction of the marine organic carbon pool. However, little is known about the possible interactions of BC and marine microorganisms. Here, we report the results of experiments using a standard reference BC material in high concentrations to investigate basic principles of the dynamics of natural bacterial and viral communities with BC particles. We assessed the attachment of viral and bacterial communities using scanning electron, epifluorescence and confocal laser scanning microscopy and shifts in bacterial community composition using 16S rRNA gene denaturing gradient gel electrophoresis (DGGE). In 24-h time-course experiments, BC particles showed a strong potential for absorbing viruses and bacteria. Total viral abundance was reduced, whereas total bacterial abundance was stimulated in the BC treatments. Viral and bacterial abundance on BC particles increased with particle size, whereas the abundances of BC-associated viruses and bacteria per square micrometer surface area decreased significantly with BC particle size. DGGE results suggested that BC has the potential to change bacterial community structure and favour phylotypes related to Glaciecola sp. Our study indicates that BC could influence processes mediated by bacteria and viruses in marine ecosystems.

Microbial production of recalcitrant dissolved organic matter: long-term carbon storage in the global ocean.

The biological pump is a process whereby CO(2) in the upper ocean is fixed by primary producers and transported to the deep ocean as sinking biogenic particles or as dissolved organic matter. The fate of most of this exported material is remineralization to CO(2), which accumulates in deep waters until it is eventually ventilated again at the sea surface. However, a proportion of the fixed carbon is not mineralized but is instead stored for millennia as recalcitrant dissolved organic matter. The processes and mechanisms involved in the generation of this large carbon reservoir are poorly understood. Here, we propose the microbial carbon pump as a conceptual framework to address this important, multifaceted biogeochemical problem.

Trade-offs between competition and defense specialists among unicellular planktonic organisms: the "killing the winner" hypothesis revisited.

A trade-off between strategies maximizing growth and minimizing losses appears to be a fundamental property of evolving biological entities existing in environments with limited resources. In the special case of unicellular planktonic organisms, the theoretical framework describing the trade-offs between competition and defense specialists is known as the "killing the winner" hypothesis (KtW). KtW describes how the availability of resources and the actions of predators (e.g., heterotrophic flagellates) and parasites (e.g., viruses) determine the composition and biogeochemical impact of such organisms. We extend KtW conceptually by introducing size- or shape-selective grazing of protozoans on prokaryotes into an idealized food web composed of prokaryotes, lytic viruses infecting prokaryotes, and protozoans. This results in a hierarchy analogous to a Russian doll, where KtW principles are at work on a lower level due to selective viral infection and on an upper level due to size- or shape-selective grazing by protozoans. Additionally, we critically discuss predictions and limitations of KtW in light of the recent literature, with particular focus on typically neglected aspects of KtW. Many aspects of KtW have been corroborated by in situ and experimental studies of isolates and natural communities. However, a thorough test of KtW is still hampered by current methodological limitations. In particular, the quantification of nutrient uptake rates of the competing prokaryotic populations and virus population-specific adsorption and decay rates appears to be the most daunting challenge for the years to come.

Assessing niche separation among coexisting Limnohabitans strains through interactions with a competitor, viruses, and a bacterivore.

We investigated potential niche separation in two closely related (99.1% 16S rRNA gene sequence similarity) syntopic bacterial strains affiliated with the R-BT065 cluster, which represents a subgroup of the genus Limnohabitans. The two strains, designated B4 and D5, were isolated concurrently from a freshwater reservoir. Differences between the strains were examined through monitoring interactions with a bacterial competitor, Flectobacillus sp. (FL), and virus- and predator-induced mortality. Batch-type cocultures, designated B4+FL and D5+FL, were initiated with a similar biomass ratio among the strains. The proportion of each cell type present in the cocultures was monitored based on clear differences in cell sizes. Following exponential growth for 28 h, the cocultures were amended by the addition of two different concentrations of live or heat-inactivated viruses concentrated from the reservoir. Half of virus-amended treatments were inoculated immediately with an axenic flagellate predator, Poterioochromonas sp. The presence of the predator, of live viruses, and of competition between the strains significantly affected their population dynamics in the experimentally manipulated treatments. While strains B4 and FL appeared vulnerable to environmental viruses, strain D5 did not. Predator-induced mortality had the greatest impact on FL, followed by that on D5 and then B4. The virus-vulnerable B4 strain had smaller cells and lower biomass yield, but it was less subject to grazing. In contrast, the seemingly virus-resistant D5, with slightly larger grazing-vulnerable cells, was competitive with FL. Overall, our data suggest contrasting ecophysiological capabilities and partial niche separation in two coexisting Limnohabitans strains.